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The crystal structure of a site-specific mutant of the N-terminal half-molecule of human lactoferrin, Lf(N), in which the iron ligand Asp60 has been mutated to Ser, has been determined at 2.05 A resolution in order to determine the effects of the mutation on iron binding and domain closure. Yellow monoclinic crystals of the D60S mutant, in its iron-bound form, were prepared, and have unit cell dimensions a = 110.2 A, b = 57.0 A, c = 55.2 A, beta = 97.6 degrees, space group C2, with one molecule of 333 residues in the asymmetric unit. The structure was determined by molecular replacement, using the wild-type Lf(N) as search model, and was refined by restrained least-squares methods. The final model, comprising 2451 protein atoms (from residues 2 to 315) one Fe3+ and one CO2-(3), and 107 water molecules, gives an R-factor of 0.175 for all data in the resolution range 20.0 to 2.05 A. The model conforms well with standard geometry, having root-mean-square deviations of 0.014 A and 1.2 degrees from standard bond lengths and angles. The structure of the D60S mutant deviates in two important respects from the parent Lf(N) molecule. At the mutation site the Ser side-chain neither binds to the iron atom nor makes any interdomain contact as the substituted Asp does; instead a water molecule fills the iron coordination site and participates in interdomain hydrogen bonding. The domain closure is also changed, with the D60S mutant having a more closed conformation. Consideration of crystal packing suggests that the altered domain closure is a genuine molecular property but both the iron coordination and interdomain contacts are consistent with weakened iron binding in the mutant. The implications for iron binding in transferrins generally are discussed.

Enzymatic deglycosylation has been used in attempts to crystallize several glycoproteins with the aim of overcoming the problems resulting from heterogeneity and flexibility of the attached glycan chains. An endoglycosidase preparation from Flavobacterium meningosepticum, comprising the enzymes endo F and PNGase-F, was used in experiments on the mammalian binding proteins lactoferrin and haemopexin. Significant differences were found in the susceptibility of different proteins to deglycosylation. For human lactoferrin (Lf) and its recombinant N-terminal half-molecule (Lf(N)), deglycosylation was rapid and complete, and was essential for obtaining high-quality crystals of both apo-Lf and Lf(N); for bovine Lf, however, complete deglycosylation did not occur. Similarly, for rabbit haemopexin the carbohydrate chain on the C-terminal domain was easily removed, but the three chains on the N-terminal domain proved more resistant and their removal led to some fragmentation of the protein. Nevertheless, this approach provided the only means of crystallizing the C-terminal domain and is likely to be useful for other glycoproteins.

The three-dimensional structures of various forms of lactoferrin, determined by high resolution crystallographic studies, have been compared in order to determine the relationship between structure and biological function. These comparisons include human apo and diferric lactoferrins, metal and anion substituted lactoferrins, the N-terminal half molecule of human lactoferrin, and bovine diferric lactoferrin. The structures themselves define the nature and location of the iron binding sites and allow anti-bacterial and putative receptor-binding regions to be mapped on to the molecular surface. The structural comparisons show that small internal adjustments can allow the accommodation of different metals and anions without altering the overall molecular structure, whereas large-scale conformational changes are associated with metal binding and release, and smaller, but significant, movements accompany species variations. The results also focus on differences in flexibility between the two lobes, and on the importance of interactions in the inter-lobe region in modulating iron release from the N-lobe and in possibly enabling binding at one site to be signalled to the other.

The full length copy DNA (cDNA) for human lactoferrin has been synthesised by the polymerase chain reaction (PCR) using sequence specific primers. The template was first strand cDNA, synthesised from human bone marrow RNA using oligo(dT) to prime DNA synthesis by MMLV reverse transcriptase. The full-length human lactoferrin cDNA has been expressed in baby hamster kidney (BHK) cells using the expression vector pNUT. The protein expressed from the cloned cDNA is secreted into the culture medium and yields of up to 40 mg per litre have been obtained. A mutant protein corresponding to the N-lobe of human lactoferrin (LfN) has also been expressed in BHK cells. The cDNA coding for this protein was produced by the introduction of stop codons into the region of the cDNA corresponding to the helix linking the N- and C-lobes of the native protein. LfN is also expressed as a secreted protein and has been obtained in high yield. LfN binds iron and has UV/Vis and ESR spectra which are virtually identical to the native protein. However, the pH at which iron is released from LfN is quite different to the pH of iron release from the native and the full-length recombinant protein. A number of mutations have been introduced into LfN by site-directed mutagenesis and the mutant proteins expressed in BHK cells. These mutations involve the iron binding ligands and have been designed to introduce some of the changes found in the C-lobe of melanotransferrin into LfN. An attempt has been made to express a protein corresponding to the C-lobe of human lactoferrin (LfC) by attaching the sequence for the signal peptide of lactoferrin to the cDNA sequences coding for the C-lobe.

The three-dimensional structure of the N-terminal half-molecule of human lactoferrin, LfN, prepared by recombinant DNA methods, has been determined by X-ray crystallography at 2.0 A resolution. The protein is in its iron-bound form and is deglycosylated. X-ray diffraction data were obtained by diffractometry to 3.2 A resolution and synchrotron data collection, using Weissenberg photography with imaging plates, to 1.8 A resolution. The structure was solved by molecular replacement, using the N-lobe of native diferric human lactoferrin (Lf) as search model. Restrained least squares refinement (program TNT) has resulted in a model structure with an R-factor of 0.184 for all data 34,180 (reflections) in the resolution range 8.0 to 2.0 A. The model comprises 2490 protein atoms (residues 4 to 327), 1 Fe3+, 1 CO3(2-) and 180 solvent molecules, all regarded as water. The structure of LfN is essentially the same as that of the N-lobe of intact Lf, being folded into two similar alpha/beta domains, with the Fe3+ and CO3(2-) bound in a specific site in the interdomain cleft. These details are not affected by either deglycosylation or expression in a non-native system. At the C terminus, however, the conformation of residues 321 to 333 is changed. Whereas in Lf residues 321 to 332 form a helix crossing between the domains at the back of the iron site, in LfN residues 321 to 326 have an extended conformation, forming a third interdomain beta-strand, and residues 328 to 333 appear disordered. The conformational change is attributed to the loss of stabilizing interactions from the C-lobe and is mediated by two Gly residues, at positions 321 and 323. It is further proposed that the conformational change is responsible for the more facile iron release properties of LfN, by its effect on the hinge mechanism and increased solvent exposure of residues near the back of the iron site. Other details of the polypeptide chain conformation and the binding site have also been analysed. Two cis-proline residues are found at positions 71 and 142. The bidentate binding of the CO3(2-) to the metal ion is unambiguous, and a network of hydrogen bonds in and around the binding site links the two domains. Clearly-defined amino-aromatic hydrogen bonds are found for Arg210, near the metal site, and some 31 internal water molecules have been identified, 15 of them in essentially discrete sites, and 16 in a cluster filling a cavity in the interdomain cleft.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.

The factors influencing iron binding and release by lactoferrin have been addressed by comparison of the native full length molecule (Lf) with the N-terminal half of human lactoferrin (LfN) produced from the cloned cDNA expressed in baby hamster kidney (BHK) cells. The coding sequences for LfN were inserted into the expression vector pNUT between the metallothionein promoter and the human growth hormone transcription termination sequences. Transformed BHK cells were grown in roller bottles where concentrations of LfN as high as 35 mg/liter were obtained. The pure protein, produced by the transformed BHK cells, was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein blotting and immunodetection, N-terminal sequence analysis, UV-visible spectroscopy, electron spin resonance spectroscopy, and measurements of metal binding and release. By these criteria LfN was found to be correctly processed, glycosylated, and able to bind iron reversibly. Both UV-visible and electron spin resonance spectra of the half molecule were very similar to those of native lactoferrin and the full length lactoferrin produced in BHK cells, but there were marked differences in the pH at which iron release occurred. Iron release from LfN occurs in the pH range 6.0-4.0, compared with 4.0-2.5 for native lactoferrin and 6.2-4.0 for transferrin. These results suggest that the more facile release of iron from LfN compared with native lactoferrin results from the absence of stabilizing contacts between the N- and C-terminal halves and that the characteristic difference in pH stability between lactoferrins and transferrins is due primarily to differences in these interactions.

We reviewed all studies since 1940 on the prognosis of squamous cell carcinoma (SCC) of the skin and lip. The following variables are correlated with local recurrence and metastatic rates: (1) treatment modality, (2) prior treatment, (3) location, (4) size, (5) depth, (6) histologic differentiation, (7) histologic evidence of perineural involvement, (8) precipitating factors other than ultraviolet light, and (9) host immunosuppression. Local recurrences occur less frequently when SCC is treated by Mohs micrographic surgery. This local recurrence rate differential in favor of Mohs micrographic surgery holds true for primary SCC of the skin and lip (3.1% vs 10.9%), for ear SCC (5.3% vs 18.7%), for locally recurrent (previously treated) SCC (10% vs 23.3%), for SCC with perineural involvement (0% vs 47%), for SCC of size greater than 2 cm (25.2% vs 41.7%), and for SCC that is poorly differentiated (32.6% vs 53.6%).

We reviewed all studies (since 1945) reporting recurrence rates for treatment of recurrent (previously treated) basal cell carcinomas (BCC) using surgical excision, radiotherapy, cryotherapy, curettage and electrodesiccation, and Mohs micrographic surgery. The 5-year recurrence rate for Mohs micrographic surgery is 5.6%. The recurrence rate for non-Mohs modalities of 19.9% is nearly four times higher. Individual recurrence rates for the non-Mohs modalities are 17.4% for surgical excision, 40.0% for curettage and electrodesiccation, and 9.8% for radiation therapy. There are no studies reporting 5-year data for cryotherapy. However, the recurrence rate is 13.0% for cryotherapy when the follow-up period is less than five years. The data support the following conclusions: (1) Mohs surgery is the treatment of choice for recurrent BCC; (2) if the patient is not a surgical candidate and the lesion is small, radiation therapy is an alternative that offers a better chance for cure than the other non-Mohs modalities; and (3) curettage and electrodesiccation should not be used to treat recurrent basal cell carcinoma.

The purposes of this study were to replicate a previous study of the relationship between alcohol consumption and cognitive functioning in college students and to investigate the reversibility of negative effects of social drinking on cognitive functioning when randomly assigned subjects abstained from drinking for 2 weeks. The previous study was replicated by administering the same battery of neuropsychological tests to 170 subjects (103 women) during the first testing session. Like the original study, the present study demonstrated several significant predicted inverse relationships between drinking and cognitive performance, but specific relationships between various drinking and cognitive variables were not replicated. As in the original study, some significant nonpredicted relationships also occurred. At the end of the first testing session, subjects were randomly assigned either to abstain from drinking or to maintain their usual drinking patterns for 2 weeks. They were then administered a different neuropsychological battery designed to assess functions similar to the original battery. Consumption the previous 2 weeks was significantly lower in the abstain group than in the maintain group, but there were no significant differences in the predicted direction between groups on the cognitive variables. Several significant predicted inverse correlations between drinking variables and cognitive performance occurred, but some nonpredicted relationships occurred also. Problems and implications for research in social drinking are discussed.

"Ulceration width" is superior to "ulceration per se" as a prognostic variable in patients with clinical stage I melanoma. Just as thickness has replaced level of invasion, "ulceration width" will replace ulceration.

We reviewed photographs of 256 primary cutaneous melanomas to determine the gross morphological correlates of metastases. Seven and a half years after diagnosis, the melanomas with ulceration occupying at least 80% of their surface had the highest rate of metastases (85%), and melanomas without a nodule had the lowest metastatic rate (11%). Melanomas with nodules had a metastatic rate of 62%, and this rate increased in direct proportion to nodule diameter. Even after adjusting for nodule diameter and ulceration, melanomas with single nodules located completely within the confines of an associated plaque had half of the metastatic rate of melanomas with nodules located at the periphery (abutting normal skin). These data suggest that (1) carefully recorded gross pathological data can augment the microscopic pathological data in the determination of prognosis; (2) skin lesions suspected to be melanoma should be photographed; (3) the photograph, if followed by surgical removal of the lesion, should be attached to the pathology report in the patient's permanent medical record; (4) nodule diameter is better correlated with metastases than the total lesion diameter (as traditionally held); and (5) the cytologically malignant melanocytes that constitute the less-raised portion of most melanomas may not be biologically malignant, thus enlarging the precursor concept for malignant melanoma. The TNM staging system for malignant melanoma could be modified to incorporate these data.

We studied 14 prognostic factors in 428 patients with clinical stage I melanoma to determine which factor or combination of factors was associated with death from melanoma from 24 to 60 months following diagnosis. Forty-eight patients (11 percent) died during this period. All 17 patients who had visceral metastases present at 24 months died during this period. All surviving patients were followed for at least 60 months. Individual high risk factors included ulceration width (as determined by histology), level IV or V tumor, recurrence other than visceral, 6 or more mitoses per square millimeter, presence of involved nodes on elective dissection, absent or slight lymphocyte response, tumor type other than superficial spreading, location other than extremities (excluding hands and feet), microscopic satellites, thickness, sex, and wide local excision. The presence of sex as a risk factor for patients dying from 2 to 5 years following diagnosis is noteworthy because no sex difference was noted in the early death (less than 24 months) group. Age, presence of a nevus, and histologic regression were not significant factors. A logistic regression analysis selected a combination of the following independent factors: (1) location on extremities excluding hands and feet (favorable), (2) thickness, (3) recurrence other than visceral, (4) positive elective nodal dissection, (5) 6 or more mitoses per square millimeter, and (6) moderate to marked lymphocyte response (favorable). Twenty-five percent of patients with level IV lesions died between 24 and 60 months compared with only a 6 percent death rate within the first 24 months.

Thirteen variables were studied for their relative usefulness in predicting recurrent disease in 107 patients with clinical Stage I melanoma of the upper extremity. After a mean follow-up period of 54 months, the only patents who have had recurrent disease to date are those who primary lesions were located either on the hand or posterior upper arm. The five-year disease-free survival role for 44 patients with melanoma at these sites was 68%. None of 63 patients with melanoma located on the forearm of anterior upper arm have had recurrent disease (i.e., the five-year, disease-free survival rate was 100% (p = 0.00004), compared with the hand or posterior arm group). A Cox proportional hazards (multivariate) analysis demonstrated that two primary tumor histologic variable, thickness in millimeters and ulceration, interacted to produce the best prognostic model for those 44 patients with melanoma of the hand or posterior upper arm. Twenty-one patients with primary lesions at these sites had primary tumors less than 2.25 mm in thickness and no evidence of ulceration histologically. Their five-year, disease-free survival role was 95%. For the remaining 23 patients with primary tumors on the hand or posterior upper arm who had either histologic evidence of ulceration or primary tumors greater than or equal to 2.25 mm, the five-year disease-free survival rate was 37% (p = 0.002, compared with group nonulcerated, thin lesions). The excellent survival rate for patients with melanomas on the forearm or anterior upper arm was not completely explained by pathologic stage, by primary tumor thickness, or by histologic ulceration of the primary tumor.

The association of T lymphocytes and dendritic cells with the stromal mononuclear cell response to basal cell carcinomas has led to speculation that cellular immunity may, in part, regulate the growth and development of this neoplasm. It has not been established, however, whether these T cells are functionally competent, or simply coincidental bystanders. We examined the immunologic phenotypes of mononuclear cells in 32 lesions of basal cell carcinoma obtained from 26 patients. The majority of infiltrating mononuclear cells were T cells that were equally distributed between the helper/inducer (Leu 3a+) and cytotoxic/suppressor (Leu 2a+) subtypes; a minority of cells were dendritic and expressed Leu 6 antigen. Virtually all T cells and dendritic cells were HLA-DR+, and many (greater than 30%) of the T cells expressed antigens consistent with stages of ongoing activation (T9, T10). TS2/7, a novel monoclonal antibody recently documented to identify activation-specific subcomponents of 210/165/130 kD glycoprotein complex present on the surface of mitogen- or alloantigen-stimulated human T cells, was also used. Greater than 50% of the T cells observed were TS2/7+. These observations provide in situ immunomorphologic evidence of stromal T cell activation in association with basal cell carcinomas, and suggest a role for active and ongoing cellular immune mechanisms as a determinant of local biological behavior of this neoplasm.

Every prospective study published to date, whether randomized or nonrandomized, shows that the survival rate for patients with clinical stage I melanoma is the same, irrespective of whether they have an elective lymph node dissection (ELND). All the studies that purport to show a survival benefit from ELND have been based on retrospective data and are therefore subject to selection bias. Nevertheless, the data support the notion that there may be a small, select group of melanoma patients whose lives can be saved by ELND. This subgroup is made up of those patients who have epithelioid in small nests (ESN) melanomas. In the early stages of its evolution, this type of malignant melanoma metastasizes to regional lymph nodes and often does not have coexistent distant metastases. Other types of melanomas, when they metastasize, either bypass the lymph nodes or metastasize to the lymph node and simultaneously send distant metastases elsewhere, nullifying the anticipated benefit from an ELND. We encourage dermatologists, when they have a choice, to be supportive of those surgeons who have a choice, to be supportive of those surgeons who are actively participating in ongoing randomized trials designed to select patients who might benefit from ELND.

We studied 48 patients with lentigo maligna melanoma (LMM) and compared the clinical stage I patients with non-LMM melanoma patients (matched by site and thickness) to see if prognosis differed. There was no significant difference in mortality from melanoma between the two groups (P = .68) after a mean follow-up time of five years (67.5 months for LMM, 60.5 months for non-LMM). In addition, a Cox multivariate analysis of the entire matched group showed that only thickness was significantly associated with death from melanoma (P = .0007) while histology (LMM v non-LMM) did not make a significant contribution (P = .61). Our data suggest that after accounting for primary tumor thickness and site, LMM and non-LMM have the same prognosis and biologic behavior, in contrast to the widely held belief that LMM has a better prognosis than other forms of melanoma.

"Mystified" best characterizes the feeling of many physicians as they read the results of a multivariate analysis. Much of the mystery disappears when two items are shown: (1) explicit tabulations revealing how each factor selected by the analysis relates to outcome, both singly and jointly with other factors; and (2) alternative models generated by repeating the analysis after removing the most important variables one by one from the list of factors that was initially analyzed. In this way, one can determine which variables are good stand-ins or substitutes for the best variables in the initial analysis. Showing these substitutes may expose exaggerations in benefits attributed to a particular therapy (ie, elective regional node dissection for clinical stage I malignant melanoma). We have applied these guidelines to examples in the literature, particularly studies of malignant melanoma.

In order to assess a relationship between small congenital nevocellular nevi and cutaneous melanoma, histologic features commonly associated with congenital nevi were sought in 234 melanomas. The detection of one or more histologic features of congenital nevi in 8.1% (19/234) of melanoma specimens was directly related to the number of slides and tissue sections with melanoma available for review, the predominance of superficial spreading melanoma (SSM) and the historic relationship to a preexisting pigmented nevus at the tumor site. The histologic association was inversely related to melanoma thickness and tumor location on the lower extremities. The observed frequency of histologic association was estimated to be approximately 4,000 to 13,000 times greater than expected on the basis of surface area by chance alone. These findings suggest that small congenital nevi may be precursors for at least some cases of cutaneous melanoma. The strength of histologic association is highly dependent on the specificity of methods used for detecting congenital nevi in melanoma specimens.

A 12-year-old girl was evaluated for a widespread eruption of Spitz nevi. Multiple Spitz nevi are rare, and this variant is exceedingly rare. The reported cases of eruptive nevi of all types are briefly reviewed. Very little is known regarding cause, natural history, or treatment. Cutaneous stimulation occurring in a genetically predisposed individual is the proposed essential causative factor in this disorder.

For post-Mohs surgical defects of the head and neck, the width of the defect is the best predictor of the length of that time it will take the wound to heal. This conclusion is based on a multivariate analysis, testing the effect of the following factors on wound-healing time: (1) age, (2) sex, (3) sound location, (4) wound width, (5) wound length, and (6) wound length X wound width. After accounting for wound width, none of the other factors significantly influenced the rate of wound healing. The 64 wounds evaluated were dressed daily with one of five bandages. When compared with the controls, and after correcting for wound width, each of the test bandages shortened wound-healing time. Among the treatment groups, no significant differences were found in the cultures or in the appearance of the wounds.

Heavy cigarette smoking (current smoking with greater than 15-pack-year smoking history), along with 13 other variables, were tested for their ability to predict death in 196 patients with clinical Stage I melanoma. A stepwise proportional hazards general linear model (Cox multivariate analysis) showed that although heavy cigarette smoking as a single variable is an adverse prognostic marker (P = 0.0065), it has only suggestive prognostic significance once thickness factors are taken into account (P = 0.0747). People who stopped smoking had the same survival as nonsmokers. Clinical Stage I patients with melanoma who were heavy smokers presented with thicker lesions than their nonsmoking counterparts (P = 0.037). Although cigarette smoking may play a role in the biologic behavior of melanoma by mediating an effect on thickness, it need not be considered as an independent stratification criterion when analyzing results of melanoma prognosis or treatment.

We studied 13 prognostic factors in 582 patients with clinical stage I melanoma to determine which factor or combination of factors was associated with death from melanoma within the first 24 months following diagnosis. Thirty-six patients died during this period. Only 2 deaths occurred in patients with primary tumors thinner than 1.70 mm, and only 2 patients of 189 died with tumors located on the non-BANS extremities, excluding the hands and feet. Individual factors associated with high risk for death within 2 years included level V tumors, acral location, thickness greater than or equal to 3.65 mm, histologic ulceration greater than 3 mm, nodular type, presence of microscopic satellites, greater than 6 mitoses/mm(2), positive elective node dissection, absence of lymphocyte response at the tumor base, and absence of an associated nevus histologically. Many of the preceding individual factors are highly correlated. By the use of logistic regression analysis, only one very high risk group was found: 71 percent of patients with level V tumors greater than 1.70 mm thick with histologic ulceration width greater than 3 mm located in an area other than the extremities (excluding hands and feet) had died within 2 years of diagnosis. The ability to select high-risk groups should be useful to investigators involved with the design and evaluation of adjuvant therapy studies.

A review of the microscope slides of the primary tumors for 596 patients with clinical Stage I melanoma revealed that primary lesions displayed two distinct patterns of invasion: 1) single cell invasion with direct extension of the main body of tumor into the reticular dermis or subcutaneous fat, and 2) invasion with "microscope satellites" (i.e. discrete tumor nests greater than 0.05 mm in diameter, that were separated from the main body of the tumor by normal reticular dermal collagen or subcutaneous fat). The five-year disease free survival rate for 95 patients with "microscopic satellites" was 36% +/- 6%. This is in contrast to a five-year disease free survival rate of 89% +/- 2% for 501 patients without these satellites (p = 4.3 x 10(-29), generalized Wilcoxon test). "Microscopic satellites" (present vs absent) was comparable to histologic ulceration in its additive prognostic effect of tumor thickness (Breslow).

We reviewed all studies (since 1947) reporting recurrence rates for treatment of primary (previously untreated) basal cell carcinomas using surgical excision, radiotherapy, cryotherapy, curettage and electrodesiccation, and Mohs micrographic surgery. Our findings indicate that recurrences following treatment of primary basal cell carcinoma appear later than is generally acknowledged in the literature. We found that less than one-third of all recurrences appear in the first year following treatment; only 50% appear within the first 2 years following treatment; and only 66%, or nearly two-thirds, appear within the first 3 years following treatment. A good rule of thumb is that the 10-year recurrence rate is double, or 2 times, that of the 2-year recurrence rate. Furthermore, 18% of recurrences appear between the fifth and tenth year following treatment. These results held true, irrespective of treatment modality examined. Seventy-two studies reporting short-term recurrence rates (follow-up less than 5 years) had a weighted average recurrence rate of 4.2%, whereas 34 long-term studies (follow-up of 5 years) had a weighted average recurrence rate of 8.7%, or more than 2 times the short-term rate. Five-year recurrence rates by treatment modality are as follows: Mohs micrographic surgery 1.0%, surgical excision 10.1%, curettage and electrodesiccation 7.7%, radiation therapy 8.7%, and cryosurgery 7.5%. We conclude that the reporting of recurrence rate data for basal cell carcinoma should be standardized using 5-year life table analysis, and even more important is our conclusion that lifetime follow-up is necessary after treatment of primary basal cell carcinoma in order both recurrences and new primaries.

Surgical resectability is the most important prognostic factor for determining the length of survival following distant metastases. Other important factors that derive prognostic significance from their correlation with surgical resectability include (1) number of metastatic sites, (2) performance status, (3) initial site of metastases, (4) interval between initial diagnosis of primary tumor and onset of distant metastases. Factors which have little or no prognostic value include location of the primary tumor, sex, age and histological characteristics of the primary tumor.

Fifteen variables were studied for their usefulness in predicting recurrent disease in 254 patients with clinical stage I melanoma of the trunk. Thickness of the primary tumor correctly predicted outcome with an accuracy of 90 percent or greater in 176 patients with melanoma primaries with a thickness of less than 1.70 mm or 5.5 mm or greater. No other variables significantly increased predictive accuracy over these ranges of thickness. A Cox proportional hazards analysis of the remaining 78 patients with primary tumors 1.70 to 5.49 mm thick demonstrated that the following four variables functioned as independent risk factors for recurrent disease: (1) thickness of the primary tumor (p = 0.0005), (2) mitoses/mm2 greater than 6 (p = 0.006), (3) a nearly absent or minimal lymphocyte response at the base of the tumor (p = 0.009), and (4) location on the upper trunk (p = 0.03). Trunk lesions located near the midline did not have a worse prognosis than more lateral melanomas of similar thickness.

Fourteen variables were tested for their prognostic usefulness in 203 patients with clinical Stage I melanoma and primary tumor 0.76-169 mm thick. Only two variables, primary tumor location and level of invasion, were useful in predicting death from melanoma for these patients. Of the 12 deaths from melanoma, 11 occurred in patients with primary tumors located on the upper back, posterior arm, posterior neck, and posterior scalp (=BANS). There has been only one death from melanoma in 136 patients with melanoma located at other sites (11/67 vs 1/136, p less than 0.0001 Fisher's Exact Test). Of the 67 BANS patients, 51 had level II or level III lesions and five (10%0 died of melanoma. This compared with six deaths from melanoma in 16 patients (37.5%) with level IV BANS lesions (5/51 vs 6/16, p = 0.01 Fisher's Exact Test). The relatively high incidence of both melanoma deaths and regional node metastases for the BANS group merits consideration for testing the efficacy of elective regional node dissection for these patients.

Thirteen variables were studied to determine their usefulness in predicting recurrent disease in 158 patients with stage I melanoma of the lower extremity. A Cox proportional hazards analysis demonstrated that three variables were independent risk factors for recurrent disease in these patients: (1) thickness, in millimeters, of the primary tumor (P = 0.000009), (2) primary tumor location on the foot (P = 0.0003), and (3) the number of mitoses/mm2 (P = 0.0244). Life-table analyses of patient subgroups defined by different combinations of these three variables demonstrated that thick (greater than or equal to 3.0 mm) melanomas of the foot were associated with recurrent disease much more frequently than tumors of similar thickness located on the thigh or calf. These data provide guidelines that can be used to evaluate results of surgical and/or adjuvant therapy studies for patients with melanoma of the lower extremity.

Dysplastic melanocytic nevi (DMN) are irregularly pigmented lesions characterized by (1) atypical melanocytic hyperplasia in a lentiginous epidermal pattern (AMHL), (2) one or more dermal mesenchymal changes, and (3) frequently a dermal nevocellular nevus. In order to determine an association between DMN and cutaneous melanoma, the dominant histologic feature of DMN (namely, AMHL) was sought in histologic contiguity with 234 primary melanomas. Of these 234 cases, 9 were lentigo maligna melanomas. Of the remaining 225 cases, 49 (21.8%) were associated with AMHL in the same histologic section as (but beyond the most lateral margin of) intraepidermal and invasive melanoma. AMHL was directly associated with the presence of dermal nevocellular nevi in histologic contiguity with melanoma, and a greater number of histologic slides with melanoma available for review. AMHL was inversely associated with nodular melanoma. Most of the AMHL cases were not associated with familial melanoma, but the total number of familial cases was low. The histologic association between AMHL and melanoma in one fifth of cases in this series supports the hypothesis that at least some cutaneous melanomas may have an origin in DMN.

Regional lymph node enlargement, as determined by palpation, is a useful prognostic factor--even if these nodes are histologically free of metastases. This is true not only for malignant melanoma but also for mycosis fungoides and carcinoma of the vulva.

Decreased cognitive performance was significantly correlated with increased quantity of alcohol per occasion and with total lifetime consumption in both women and men college students tested while sober. In men, however, increased performance on some tasks was also significantly correlated with increased frequency of drinking.

Fourteen variables were tested for their ability to predict visceral or bony metastases in 177 patients with clinical Stage I melanoma of intermediate thickness (1.51 - 3.39 mm). A Cox multivariate analysis yielded a combination of four variables that best predicted bony or visceral metastases for these patients: 1) mitoses greater than 6/min 2 (p = 0.0007), 2) location other than the forearm of leg) p = 0.009, 3) ulceration width greater than 3 mm (p = 0.04), 4) microscopic satellites (p = 0.05). The overall prognostic model chi square was 32.40 with 4 degrees of freedom (p less than 10 (-5). Combinations of the above variables were used to separate these patients into at least two risk groups. The high risk patients had at least a 35% or greater chance of developing visceral metastases within five years, while the low risk group had greater than an 85% chance of being disease free at five years. Criteria for the high risk group were as follows: 1) mitoses greater than 6/mm 2 in at least one area of the tumor, irrespective of primary tumor location, or 2) a melanoma located at some site other than the forearm or leg and histologic evidence in the primary tumor of either ulceration greater than 3 mm wide or microscopic satellites. The low risk group was defined as follows: 1) mitoses less than or equal to 6/mm 2 and a location on the leg or forearm, or 2) mitoses less than or equal to 6/mm 2 and the absence in histologic sections of the primary tumor of both microscopic satellites and ulceration greater then 3 mm wide. The number of patients in this series who did not undergo elective regional node dissection (N = 47) was probably too small to detect any benefit from this procedure. Based on survival rates from this and other studies, it is estimated that approximately 1500 patients with clinical Stage I melanoma of intermediate thickness in each arm of a randomized clinical trial would be needed to detect an increase in survival rates from elective regional node dissection.

Two studies of the relationship between alcohol consumption and cognitive functioning in men and women college students are presented. Study 1 showed several predicted relationships of decreased cognitive performance on various tests with increased quantity of alcohol per occasion and total lifetime consumption in both women and men. Study 2a was designed to replicate study 1, but the pattern of relationship of cognitive and consumption variables was quite different, e.g., increased cognitive performance was associated with increased quantity per occasion for several tests in males. Study 2b was designed to demonstrate reversibility of the negative effects of consumption on cognition by randomly assigning half of the subjects to abstain for two weeks. Reversibility was not demonstrated. Difficulties in studying these effects in college students are discussed.

Narrow margins of excision for melanomas less than 0.85 mm thick neither shorten the length of survival nor increase the local recurrence rate. Similarly, narrow margins of excision for melanomas greater than 0.85 mm thick do not adversely affect the survival rate. However, for thicker melanomas, the local recurrence rate increases when the surgical margin is reduced to less than 3 cm.

Fourteen prognostic factors were examined in 79 patients with clinical Stage I melanoma greater than or equal to 3.65 mm in thickness. All nine patients with melanoma of the hands or feet died of melanoma. A Cox proportional hazards (multivariate) analysis of the remaining 70 patients showed that a combination of the following four variables best predicted bony or visceral metastases: 1) a nearly absent or minimal lymphocyte response at the base of the tumor, 2) histologic type other than superficial spreading melanoma, 3) location on the trunk, and 4) positive nodes or no initial node dissection. Ulceration and/or ulceration width were not useful in predicting outcome either singly or in combination with other variables. Patients with negative lymph nodes and primary tumors of the trunk, hands, and feet did not do better than patients with positive nodes at those sites. Conversely, non of 16 patients with negative lymph nodes and extremity melanomas (excluding the hands and feet) or head and neck melanomas developed visceral or bony metastases (i.e., five-year disease-free survival rate 100%).

In an earlier study we found that patients with clinical Stage 1 and 2 cutaneous malignant melanoma and increased splenic radiocolloid uptake had more frequent recurrence at 24 mo, compared with melanoma patients having normal liver-spleen scintigrams. This report, an 80-mo follow-up study, gives further information on 119 clinical Stage 1 patients. Fifteen of 35 patients with increased splenic uptake (42.9%) died from melanoma as opposed to only 16 of 84 (19.1%) with normal liver-spleen images (p less than 0.01). Multivariate analysis showed that augmented splenic uptake of technetium-99m sulfur colloid is a marker for adverse prognosis in patients with malignant melanoma but does not appear to be an independent variable in predicting death. In clinical Stage 1 patients, increased splenic uptake correlated significantly with pathologic stage (positive elective node biopsy) as well as thickness and mitotic rate in patients with thicker lesions. It may be that patients with thicker, pathologically aggressive tumors have an increased splenic blood flow and/or enhanced immune and reticuloendothelial response (as manifested by abnormal liver-spleen scintigram). If so, the enhanced immune response does not appear to contribute to overall survival.

We tested 12 clinical and histologic variables to see which ones best predicted death from melanoma in 66 patients with positive elective regional node dissections (clinical stage I, pathologic stage II [CSI, PSII]). Despite the presence of lymph node metastases, not all patients had poor prognoses. Patients with tumors less than or equal to 3.5 mm and a percentage of positive nodes less than or equal to 20% had a 7-year survival rate of 66%. Within this low-risk group the subset with primary lesions on the trunk or extremities (except hands and feet) had a 7-year survival rate of 76%. This compares with poor 7-year survivals of 29% and 30% observed in other defined high-risk groups. Our results confirm and extend earlier observations concerning the prognoses of CSI, PSII melanoma patients and are relevant to any ongoing and future studies concerning elective regional node dissection (ERND) or adjuvant therapy trials in melanoma.

Malignant melanoma is a more heterogeneous neoplasm than indicated by the four histologic types described by Clark and his associates. In this study, we focused attention on the histologic morphology of malignant melanocytes in nodules of malignant melanoma and have identified at least four additional subtypes. Determination of "nodule type" appears to be superior to the Breslow measurement of thickness in predicting metastases and death. Based on our experience with this study, we believe that a review of the microscopic slides from a larger series of cases, with at least eight years follow-up, may reveal other "nodule types" and may also further divide these four subtypes into yet more groups.